Terpenoids or terpenes represent a family of natural products found in most organisms (bacteria, fungi, animal, plants). Terpenoids are made up of five carbon units called isoprene units. They can be classified by the number of isoprene units present in their structure: monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes (C40) and polyterpenes (Cn). The plant kingdom contains the highest diversity of monoterpenes and sesquiterpenes.
The monoterpenes and sesquiterpenes are the most structurally diverse isoprenoids. They are usually volatile compounds and are mostly found in plants were they play a role in defense against pathogens and herbivores attacks, in pollinator attraction and in plant-plant communication.
Some plants, known as aromatic plants or essential-oil-plants, accumulate large amounts of monoterpenes and sesquiterpenes in their leaves. In these plants, the terpenes are often synthesized and accumulated in specialized anatomical structures, glandular trichomes or secretory cavities, localized on the leaves and stems surface. Classical examples of such plants are members from the Lamiaceae family such as lavender, mint, sage, basil and patchouli.
Monoterpene and sesquiterpene accumulating plants have been of interest for thousands of years because of their flavor and fragrance properties and their cosmetic, medicinal and anti-microbial effects. The terpenes accumulated in the plants can be extracted by different means such as steam distillation that produces the so-called essential oil containing the concentrated terpenes. Such natural plant extracts are important components for the flavor and perfumery industry.
Many sesquiterpene compounds are used in perfumery (e.g. patchoulol, nootkatone, santalol, vetivone, sinensal) and many are extracted from plants. The price and availability of the plant natural extracts is dependent on the abundance, the oil yield and the geographical origin of the plants. Because of the complexity of their structure, production of individual terpene molecules by chemical synthesis is often limited by the cost of the process and may not always be chemically or financially feasible. The recent progress in understanding terpene biosynthesis in plants and the use of modern biotechnology techniques opens new opportunities for the production of terpene molecules. The use of biocatalysts for the production of terpenes requires a clear understanding of the biosynthesis of terpenes and the isolation of the genes encoding enzymes involved in specific biosynthetic steps.
The biosynthesis of terpenes in plants has been extensively studied. The common five-carbon precursor to all terpenes is isopentenyl pyrophosphate (IPP). Most of the enzymes catalyzing the steps leading to IPP have been cloned and characterized. Two distinct pathways for IPP biosynthesis coexist in the plants. The mevalonate pathway is found in the cytosol and endoplasmic reticulum and the non-mevalonate pathway (or deoxyxylulose (DXP) pathway) is found in the plastids. In the next step IPP is repetitively condensed by prenyl transferases to form the acyclic prenyl pyrophosphate terpene precursors for each class of terpenes, e.g. geranyl-pyrophosphate (GPP) for the monoterpenes, farnesyl-pyrophosphate (FPP) for the sesquiterpenes, geranylgeranyl-pyrophosphate (GGPP) for the diterpenes. These precursors serve as substrate for the terpene synthases or cyclases, which are specific for each class of terpene, e.g. monoterpene, sesquiterpene or diterpene synthases. Terpene synthases catalyze complex multiple step cyclizations to form the large diversity of carbon skeleton of the terpene compounds. The reaction starts with the ionization of the diphosphate group to form an allylic cation. The substrate undergoes then isomerizations and rearrangements that are controlled by the active site of the enzyme. The product can be acyclic, or cyclic with one or multiple rings. The reaction is terminated by deprotonation of the carbocation or by capture by a water molecule and the terpene hydrocarbon or alcohol is released. Some terpene synthases produce a single product, but most of them produce multiple products. These enzymes are responsible for the extremely large number of terpene skeletons. Finally, in the last stage of terpenoid biosynthesis, the terpene molecules may undergo several steps of secondary enzymatic transformations such as hydroxylations, isomerisations, oxido-reductions or acylations, leading to the tens of thousand of different terpene molecules.
This invention relates to the isolation of nucleic acids encoding for sesquiterpene synthases. The sesquiterpene synthases convert farnesyl pyrophosphate to the different sesquiterpene skeletons. Over 300 sesquiterpene hydrocarbons and 3000 sesquiterpenoids have been identified (Joulain, D., and König, W. A. The Atlas of Spectral Data of Sesquiterpene Hydrocarbons, EB Verlag, Hamburg, 1998; Connolly, J. D., Hill R. A. Dictionary of Terpenoids, Vol 1, Chapman and Hall (publisher), 1991), and many new structures are identified each year. There is virtually an infinity of sesquiterpene synthases present in the plant kingdom, all using the same substrate but having different product profiles.
Several sesquiterpene synthase encoding cDNA or genes have been cloned and characterized from different plant sources, e.g., 5-epi-aristolochene synthases form Nicotiana tabacum (Facchini, P. J. and Chappell, J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11088-11092.) and from Capsicum annum (Back, K., et al. (1998) Plant Cell Physiol. 39 (9), 899-904), a vetispiradiene synthase from Hyoscyamus muticus (Back, K. and Chappell, J. (1995) J. Biol. Chem. 270 (13), 7375-7381), a (E)-β-farnesene synthases from Mentha pipperita and Citrus junos (Crock, J., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94 (24), 12833-12838; Maruyama et al (2001) Biol. Pharm. Bull. 24(10), 1171-1175), a δ-selinene synthase and a γ-humulene synthase from Abies grandis (Steele, C. L., et al. (1998) J. Biol. Chem. 273 (4), 2078-2089), δ-cadinene synthases from Gossypium arboreum (Chen, X. Y., et al. (1995) Arch. Biochem. Biophys. 324 (2), 255-266; Chen, X. Y., et al. (1996) J. Nat Prod. 59, 944-951.), a E-α-bisabolene synthase from Abies grandis (Bohlmann, J., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95 (12), 6756-6761.), a germacrene C synthase from Lycopersicon esculentum (Colby, S. M., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95 (5), 2216-2221.), an epi-cedrol synthase and an amorpha-4,11-diene synthase from Artemisia annua (Mercke, P., et al. (1999) Arch. Biochem. Biophys. 369 (2), 213-222; Mercke, P., et al. (2000) Arch. Biochem. Biophys. 381 (2), 173-180.), a germacrene D synthase from Lycopersicon esculentum (van der Hoeven, R. S., Monforte, A. J., Breeden D., Tanksley, S. D., and Steffens J. C. (2000) The Plan cell 12, 2283-2294) and germacrene A synthases from Lactuca sativa, from Cichorium intybus and from Solidago canadensis (Bennett, M. H., et al. (2002) Phytochem. 60, 255-261; Bouwmeester, H. J., et al. (2002) Plant Physiol. 129 (1), 134-144; Prosser I, et al. (2002) Phytochem. 60, 691-702).
One embodiment of the present invention relates to the isolation from patchouli plants of nucleic acid encoding for sesquiterpenes synthases. Patchouli oil is an important perfumery raw material obtained by steam distillation of the leaves from the plant Pogostemon cablin (patchouli), a Lamiaceae growing in tropical regions. The oil, which has a long-lasting pleasant odor with woody, earth and camphoraceous notes, is largely used in perfumery. In patchouli plants the biosynthesis and storage of the oil is associated with anatomically specialized structures: glandular structures found on the leaf surface and internal structures found all over the plant. The biosynthesis of the oil occurs in the early stage of the leaf development (Henderson, W., Hart, J. W., How, P, and Judge J. (1969) Phytochem. 9, 1219-1228). The oil is rich in sesquiterpenes. The sesquiterpene patchoulol (FIG. 1) is the major constituent (5 to 40%) and contributes considerably to the typical note.
The Biosynthesis of patchoulol in Patchouli (Pogostemon cablin) leaves has been studied and elucidated. Croteau and co-worker studied the mechanism of biosynthesis of patchoulol using patchouli leaf extracts and achieved the purification and characterization of the patchoulol synthase (Croteau et al (1987) Arch. Biochem. Biophys. 256(1), 56-68; Munck and Croteau (1990) Arch. Biochem. Biophys. 282(1), 55-64). A single sesquiterpene synthase is responsible for the biosynthesis of patchoulol from farnesyl pyrophosphate. The patchoulol synthase from patchouli is a multiple product enzyme synthesizing patchoulol as a main product and several secondary products including α-bulnesene, α-guaiene, α-patchoulene, β-patchoulene (FIG. 1) (Croteau et al (1987) Arch. Biochem. Biophys. 256(1), 56-68; Munck and Croteau (1990) Arch. Biochem. Biophys. 282(1), 55-64). The chemical synthesis of patchoulol and structurally related compounds involves a large number of steps and so far, there is no commercially interesting chemical process. Therefore, a biochemical route for the production of patchoulol would be of great interest. The engineering of a biochemical route for the production of Patchoulol requires the isolation of the genes encoding for patchoulol synthase.
One embodiment of the present invention provides nucleic acids isolated from patchouli leaves and encoding for sesquiterpene synthases. Another embodiment of the invention relates to the transformation of bacteria with the isolated nucleic acids of the invention, including the production of the resultant recombinant sesquiterpene synthases. For example, one embodiment of the invention relates to the use of a recombinant sesquiterpene synthase to produce a mixture of sesquiterpenes, with patchoulol being the major product. Other embodiments of the invention relate to the use of another recombinant sesquiterpene synthases to produce γ-curcumene as major product, and other recombinant sesquiterpene synthases to produce germacrane-type sesquiterpenes (FIG. 1). A further embodiment of the invention relates to the use of sesquiterpene synthases in vivo to produce at least one terpenoid, for example patchoulol.